Immortal cell line derived from grouper Epinephelus coioides and its applications therein

ABSTRACT

The present invention describes (1) an immortal cell line derived from grouper and a method for establishing the cell line; (2) methods for mass producing and purifying aquatic viruses using the immortal cell line from grouper; (3) an anti-NNV antibody and a method for producing the anti-NNV antibody; and (4) a vaccine of NNV and a method for protecting fish against NNV infection. The present immortal cell line is derived from the grouper and is susceptible to the viral families of Birnaviridae such as Infectious Pancreatic Necrosis Virus (IPNV); Herpesviridae such as Eel Herpes Virus Formosa (EHVF); Reoviridae such as Hard Clam Reovirus (HCRV); and Nodaviridae such as Nervous Necrosis Virus (NNV).

RELATED APPLICATION

This application claims the priority of U.S. Provisional PatentApplication No. 60/110,699, filed on Dec. 3, 1998, and is a division ofU.S. patent application Ser. No. 09/450,696, filed on Nov. 30, 1999,which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an immortal cell line (GF-1) derivedfrom the fin tissue of grouper Epinephelus coioides and the method ofestablishing the GF-1 cell line. The GF-1 cell line is susceptible to anumber of aquatic viruses, including, but not limited to, InfectiousPancreatic Necrosis Virus (IPNV), Eel Herpes Virus Formosa (EHVF), andNervous Necrosis Virus (NNV). This invention also relates to the methodof mass producing and purifying the aquatic viruses using an immortalcell line from grouper such as the GF-1 cell line as a host.Additionally, this invention relates to an anti-NNV antibody and themethod of producing the anti-NNV antibody. Finally, this inventionrelates to a vaccine of NNV and the method for protecting fish againstNNV infection.

BACKGROUND OF THE INVENTION

Nervous necrosis virus (NNV), a pathogen found in many varieties ofhatchery-reared marine fish, has caused mass mortality of such fish attheir larval or juvenile stages. NNV belongs to the family Nodaviridae.Fish nodaviruses isolated from different species (such as SJNNV, BFNNV,JFNNV, TPNNV, RGNNV, GNNV etc.) are closely related to each other owingto the high similarity of the conserved region of their coat proteingenes. NNV, also named as fish encephalitis virus (FEV) and piscineneuropathy nodavirus (PNN), is an unenveloped spherical virus withparticles sized between 25 and 34 nm. The virus is characterized byvacuolation of the nerve tissues. Viral Nervous Necrosis (VNN) diseasehas been found in many countries under various names such as viral fishencephalitis, fish encephalomyelitis, cardiac myopathy syndrome. Thehosts of NNV include many species of marine fish, for example;parrotfish, sea bass, turbot, grouper, stripped jack, tiger puffer,berfin flounder, halibut, barramundi, and spotted wolffish.

According to the statistics shown in 1993, approximately 159 fish celllines have been established which have demonstrated a capacity forgrowing fish viruses (Fryer and Lannan, J. Tissue Culture Method (1994),10:57-94). Most of these cell lines are derived from the tissues offreshwater fish. There are only thirty-four cell lines which areoriginated from marine fish. Although some of the fish cell lines, whichinclude RTG-2, CHSE-214, BF2, SBL, FHM, EPC, have been tested for thesusceptibility of fish nodavirus, none of these cells lines has showncytopathic effects (CPE) after viral inoculations.

In 1996, SSN-1 cell line, a cell line derived from striped snakeheadChanna Striatus, has been successfully used for isolating sea bassnodavirus (Frerichs et al., J. General Virology (1996)77:20672071).However, SSN-1 cell line has been known to be persistently contaminatedwith C-type retrovirus (Frerichs et al., J. General Virology (1991)72:2537-2539). Therefore, it is not suitable for the production of fishnodavirus.

Viral diseases cannot be cured by therapeutic reagents. The best ways tocontain viral diseases include prevention through early detection andthe development of vaccines. In either way, the understanding of thebiological, biochemical, and serological characteristics of the virus isfundamentally required, which in turn relies on the industry to have thecapacity of mass producing the pure form of viruses, preferably throughan in vitro cell culture system. Therefore, the development of a newcell line which can be susceptible to fish nodavirus is desperately indemand in order to control the wide spread of fish viral diseases due tofish nodavirus infection.

Grouper is an important hatchery fish in Taiwan. In recent years, therehave been several reports regarding the establishment of cell linesderived from grouper. For example, Chen et. al. (Japan ScientificSociety Press (Tokyo) (1988) 218-227) have reported their establishmentof several cell lines from the fin and kidney tissues of grouperEpinephelus awoara. Lee (Master Thesis from the Department of Zoology atthe National Taiwan University, 1993) also has reported hisestablishment of the cell lines derived from the eye pigment cells andbrain tissue of grouper Epinephelus amblycephalus. However, Chen et al.do not provide sufficient data in support of the claim for immortalityin their cell lines and Lee expressly indicates in his thesis that hisgrouper cell lines are not immortal. Moreover, neither Chen et al.'s norLee's cell lines are susceptible to fish nodavirus.

Recently, severe mortality among groupers has repeatedly occurred whichis caused primarily by nodavirus. As present, fish nodavirus has beendiscovered in grouper and can be isolated from moribund grouper whichpossess symptoms of VNN disease (Chi et al., J. Fish Disease (1997)20:185-193). Electron microscopic examination of the tissues fromgrouper shows that, in addition to nodavirus infection, grouper issusceptible to other viral infections (Chi, COA Fisheries Series No.61,Reports on Fish Disease Research (1997) 18:59-69). The fact that someviruses have host specificity makes a cell line derived from groupermore appropriate for investigating the specific viruses isolated fromgrouper.

In the invention to be presented below, an immortal cell line derivedfrom the fin tissue of grouper Epinephelus coioides (Hamilton) will beintroduced: The cell line of the present invention is susceptible tovarious viruses, particularly fish nodavirus such as GNNV. Using thepresent cell line, various aquatic viruses can be mass produced andpurified. The purified viruses are useful for antibody and vaccineproduction to protect fish from viral infections.

SUMMARY OF THE INVENTION

A first embodiment of the present invention provides for an immortalcell line derived from grouper, preferably, an immortal cell line (GF-1)which is derived from the fin tissue of grouper Epinephelus coioides.GF-1 is susceptible to, and can mass produce viruses which include, butare not limited to, viruses from the families of Birnaviridae (such asinfectious pancreatic necrosis virus [IPNV]), Herpesviridae (such as eelherpes virus Formosa [EHVF]), Reoviridae (such as hard clam reovirus[HCRV]), and Nodaviridae (such as grouper nervous necrosis virus[GNNV]).

The first embodiment also provides for a method of establishing animmortal cell line. The method comprises the steps of: (1) establishinga primary cell culture by placing cells released from the fin tissue ofgrouper Epinephelus coioides in a tissue culture flask to form amonolayer of cells; (2) subculturing and maintaining the monolayer ofcells in a media suitable for cell subculturing; and (3) monitoring atransformation of cells which is characterized by a change in chromosomenumber distribution, plating efficiency, fetal bovine serum (FBS)requirement, and susceptibility to aquatic viruses, particularly fishnodavirus such as GNNV.

A second embodiment of the invention provides for a method for growing avirus using the immortal cell line derived from grouper, preferably theGF-1 cell line. The method comprises the steps of. (1) inoculating thevirus into the cell line; and (2) incubating the cell line in a nutrientmedium suitable for growth and replication of the virus. The viruseswhich are susceptible to and can be replicated in the immortal cell lineinclude viruses from the families of Birnaviridae, Herpesviridae,Reoviridae, and Nodaviridae, and, in particular, lPNV of Birnaviridae,EHVF of Herpesviridae, HCRV of Reoviridae, and GNNV of Nodaviridae.

The second embodiment also provides for methods of mass producing theviruses using the immortal grouper cell line, purifying the viruses, anddetecting the viruses in the cell line. The method for mass producingthe virus comprises: (1) inoculating the virus into the grouper cellline; (2) incubating the cell line in a nutrient medium suitable forgrowth and replication of the virus; and (3) harvesting the virus fromthe cell line.

The method for purifying a virus comprises: (1) inoculating the virusinto the grouper cell line; (2) incubating the cell line in a nutrientmedium suitable for growth and replication of the virus until theappearance of cytopathic effects (CPE); (3) harvesting the virus fromthe cell line; and (4) purifying the virus using density gradientcentrifugation. The preferable density gradient is a CsCl densitygradient. However, other density gradients which yield a sufficientvirus harvest are also within the scope of the invention.

The present method for detecting a virus in the immortal grouper cellline comprises: observing a development of cytopathic effects (CPE) inthe cell line under microscope. The virus can be further confirmed bythe electron microscopic method which comprises the steps of. (1) fixingthe cell line in glutaraldehyde and osmium tetraoxide; (2) performingultrathin sectioning of the fixed cells; and (3) detecting viralparticles in the ultrathin section of the fixed cell line under anelectron microscope.

There are four methods which contribute to the specific detection of NNVin the immortal grouper cell line after the CPE is detected in the cellline. A first method uses the polymerase chain reaction (PCR), whichcomprises the steps of: (1) extracting a viral RNA from the cell line;(2) amplifying the viral RNA by PCR using a reverse primer (SEQ IDNO. 1) and a forward primer (SEQ ID NO.2). A second method uses awestern immunoblot, which comprises the steps of. (1) extracting theviral protein from the cell line; (2) electrophoresizing the viralprotein in an SDS-polyacrylamide gel; and analyzing the polyacrylamidegel by western immunoblot using an anti-NNV serum. A third method usesan enzyme-linked immunosorbent assay (ELISA) method to detect NNVprotein. A fourth method uses immunofluorescent staining by couplingfluorescein isothiocyanate (FITC) conjugated goat anti-mouse antibodieswith mouse anti-NNV serum to thereby detect NNV within the cells.

A third embodiment of the invention provides for an anti-NNV antibodyand a method of making the anti-NNV antibody. The anti-NNV antibody ispreferably a monoclonal antibody. The anti-NNV antibody is prepared byadministering an effective amount of NNV to a suitable animal,preferably a mouse or a rabbit, to stimulate an immunoresponse in theanimal. The NNV used for making the anti-NNV antibody is preferably theone harvested from the grouper cell line and further purified by a CsCldensity gradient centrifugation (NNV has a buoyant density ofapproximately 1.34 g/ml in CsCl).

A fourth embodiment of the invention provides for a vaccine to NNV and amethod for protecting fish against NNV infection. The NNV vaccinecomprises an immunogenically effective amount of killed NNV. The vaccinefurther comprises a material selected from the group consisting ofadjuvants, plasticizers, pharmaceutical excipients, diluents, carriers,binders, lubricants, glidants and aesthetic compounds, and combinationsthereof. The vaccine can be administered orally or by injection. Oraladministration is the preferred method of vaccinating fish. For theorally administered vaccine, an enteric coating which is impervious todissolution in the stomach of fish can be added to the vaccine. The NNVuseful for making the anti-NNV antibody is preferably the one harvestedfrom the grouper cell line and further purified by a CsCl densitygradient centrifugation (NNV has a buoyant density of approximately 1.34g/ml in CsCl). The NNV is preferably inactivated. The present method forprotecting fish against NNV infection comprises: administrating aneffective amount of the NNV vaccine to a fish.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the morphology of the GF-I cells observed under an invertedmicroscope. (A) A semi-confluent monolayer where both fibroblast-likeand epithelial cells co-existed, and (B) a confluent monolayer of GF-1cells at subculture 80 where fibroblast-like cells were the predominantcells. In the figure, an arrowhead indicates fibroblast-like cells.Similarly, an arrow indicates epitheloid cells. Bar=10 μm

FIG. 2 shows the chromosome number distribution of the GF-1 cells at (A)subculture 50, and (B) subculture 80.

FIG. 3 shows the effect of fetal bovine serum (FBS) on the growth rateof GF-1 cells at (A) subculture 50, and (B) subculture 80.

FIG. 4 shows the effect of temperature on the growth rate of GF-1 cellsat subculture 80.

FIG. 5 shows the cytopathic effects (CPE) of GF-1 cells at subculture 80after infection by (A) IPNV AB strain, (B) IPNV SP strain, (C) IPNVVR299 strain, (D) IPNV EVE strain, (E) HCRV, (F) fish nodavirus_GNNVisolate, and (G) HEVF, as compared with (H) Uninfected GF-1 cells.

FIG. 6 shows the agarose gel electrophoresis of the product by RT-PCRamplification using a pair of primers (SEQ ID NO: 1 and SEQ ID NO:2)specific to the target region T4 of fish nodavirus SJNNV. Lane 1, PCRproduct from GNNV-infected GF-1 cells; lane 2, PCR product fromnon-infected GF-1 cells. M: pGEM marker.

FIG. 7 is an electron micrograph of GNNV-infected GF-1 cells. Inclusionbodies (indicated by arrowhead) and numerous non-enveloped viralparticles are shown in the cytoplasm. I: an inclusion body filled withviral particles. M: mitochondria, N: nucleus. Arrowhead indicates theviral particle. Bar=1 μm.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with a first embodiment of the present invention, there isprovided an immortal cell line which is derived from grouper, preferablyfrom the grouper Epinephelus coioides, and more preferably from the fintissue of grouper Epinephelus coioides. A vital sample of the immortalcell line derived from the fin tissue of grouper Epinephelus coioides,the GF-1 cell line, was deposited at the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, onOct. 20, 1999, in compliance with the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purpose of Patent Procedure. The assigned deposit number for thiscell line is ATCC No. PTA-859. The viability of the GF-1 cell line wastested and confirmed on Nov. 1, 1999. The strain will be made availableif a patent office signatory to the Budapest Treaty certifies one'sright to receive, or if a U.S. Patent is issued citing the strain, andATCC is instructed by the United States Patent & Trademark Office or thedepositor to release the strain.

This embodiment also provides for a method of establishing an immortalcell line. The experimental designs and results pertaining to theestablishment of an immortal cell line are illustrated, but not limitedto, in the following examples:

EXAMPLE 1 Establishment of the GF-1 Cell Line

The GF-1 cell line was established and maintained as follows:

(1) Primary Culture

A grouper (Epinephelus coioides, Hamilton) weighing 1 kg was used forthe establishment of the primary culture. The fish was dipped in 5%chlorex for 5 min, and then wiped with 70% alcohol. The fin wasdissected from the body, and washed three times in a washing medium(containing L15 plus 400 IU/ml of penicillin, 400 μg/ml of streptomycinand 10 μg/ml of fungizone). After washing, the fin tissue was mincedwith scissors and then placed into 0.25% trypsin solution (0.25% trypsinand 0.2% EDTA in phosphate-buffered saline [PBS]). The tissue fragmentsin trypsin solution were slowly agitated with a magnetic stirrer at 4°C. At 30 min intervals, cells released from the tissue fragment werecollected by centrifugation. Next, cells were re-suspended in a completemedium (containing L15 plus 20% of fetal bovine serum [FB-S], 100 IU/mlof penicillin, 100 μg/ml of streptomycin, and 2.5 μg/ml of fungizone),transferred into a 25 cm² tissue culture flask and, finally, cultured at28° C.

(2) Subculture and Maintenance

When the confluent monolayer of cells had formed in the primary culture,cells were dislodged from the flask surface by treating with 0.1%trypsin solution (containing 0.1% of tryspin and 0.2% of EDTA in PBS).The released cells were then transferred into two new flasks containingfresh L15 medium plus 20% of FBS. Cells were subcultured at a splitratio of 1:2. For the first ten subcultures of the GF-1 cells, aconditioned medium consisting of 50% old and 50% fresh medium was used.The concentration of FBS in the maintaining L15 medium was 10% forsubcultures 11-70, and decreased to 5% after subcultures 70. Also,during the first twenty passages, GF-1 cells were subcultured at a 9-dayinterval. For the next 21^(th)-70^(th) passages, the GF-1 cells weresubcultured at a 5-day interval. After 71 passages, GF-1 cells weresubcultured at a 3 -day interval.

(3) Test for Mycoplasma Contamination in the GF-1 Cell line

The GF-1 cell line was propagated for three transfers in antibiotic-freeL15-10% FBS and tested for the presence of bacteria, fungi, andmycoplasma. A mycoplasma stain kit (Flow Laboratories, U.S.A.) was usedfor mycoplasma testing.

(4) Test for the Viability of the GF-1 Cell Line

The viability of the GF-1 cell line was tested by first removing thecells from the flask. Then, the cells were separated from the medium bycentrifugation, and re-suspended in a freezing medium consisting of 10%dimethyl sulfoxide (DMSO) and 90% FBS. Ampules (NUNC, Denmark)containing 5×10⁶ cells/ml/ampule were held at −20° C. for one hour,followed by staying at −70° C. overnight before being transferred toliquid nitrogen (−176° C.). After one month and one year, the ampuleswere thawed in a 30° C. water bath. The cells were separated from thefreezing medium by centrifugation. The cells were re-suspended inL15-10% FBS. The viable cells were determined by trypan blue staining.The number of cells was counted using a hemacytometer. The thawed cellswere re-seeded into a 25 cm² flask for further observation.

(5) Chromosome Number Distribution

The distribution of the chromosome numbers in GF-1 cells at subculture50 and subculture 80 were studied using semi-confluent and activelygrowing cells. Cells were pre-treated with 0.1 μg/ml Colcemid (Gibco,Grand Island, N.Y.) for 5 hours at 28° C. before being dislodged with0.1% of trypsin solution. After centrifugation at 1000 g for 10 min, thecells were re-suspended in a hypotonic solution (containing 8 parts ofdistilled water and 1 part of PBS) for 30 min. The cells were thenpartially fixed by adding several drops of Carnoy fixative (containing 1part of Glacial acetic acid and 3 parts of 100% methanol). The partiallyfixed cells were further centrifuged at 800 g for 10 min at 4° C. Thesupernatant was discarded, and the cells were fixed in fresh, coldCarnoy fixative for 20 min. The suspension of fixed cells was droppedonto a 76×26 mm slide. The slide was air-dried and the cells werestained with 0.4% Giemsa stain (Sigma, St. Louis, Mo., USA) for 30 min.The chromosome numbers were observed and counted under an Olympus Vanoxmicroscope.

(6) Plating Efficiency

The plating efficiency of the GF-1 cells was estimated at subcultures 50and 80. Cells were seeded into a 25 cm² flask at a density of 100 cellsper flask. Following 15 days of incubation, the medium was removed andthe cell colonies were fixed with 70% ethanol and stained with 0.4%Giemsa. The colonies in each flask were then counted using an Olympus IMinverted microscope. Carp fin (CF), black porgy spleen (BPS-1), tilapiaovary (TO-2) and eel kidney (EK) cell lines were plated the same way asthe GF-1 cell line for comparison purpose.

(7) Effects of FBS Concentration and Temperature on the Growth of theGF-1 Cells

The effects of the concentration of FBS on GF-1 cell growth weredetermined at subcultures 50 and 80. Two replicates were prepared foreach FBS concentration. At selected intervals, two flasks were withdrawnfrom each concentration of FBS, and the mean number of cells wascounted.

To determine the effects of temperature on the growth of GF-1 cells atsubculture 80, replicated cell cultures in 25 cm² flasks containingL15-10% FBS were incubated at 18° C., 28° C. and 35° C. The mean numberof the GF-1 cells from two replicated flasks at each temperature wascounted at selected intervals.

Results

Primary Culture and Subculture of the GF-1 Cells

A monolayer of cells was formed in the primary culture approximately twoweeks after the implantation. Fibroblast-like cells and epitheloid cellsco-exist in the cell population (FIG. 1). The GF-1 cells have beensuccessfully subcultured for more than 160 times since 1995,subsequently becoming a continuous cell line.

The GF-1 cells were subcultured at 9-day intervals in L15-20% of FBSduring the first twenty subcultures, at 5-day intervals in L15-10% ofFBS during the 21^(st)-70^(th) subcultures, and at 3-day intervals inL15-5% of FBS since subculture 71. Contact inhibition of the GF-1 cellswas found in cultures before subculture 50, and gradually decreasedbetween subculture 51 and 80.

The viability of the GF-1 cells at subculture 80 after one year and onemonth was 73%. The re-seeded cells grew readily when incubated at 28° C.in L15-5% of FBS.

Chromosome Number

The chromosome number of the GF-1 cells at subculture 50 was distributedbetween 7 and 44 with the mode set at 32 (FIG. 2A). The chromosomenumber of the GF-1 cells at subculture 80 was distributed between 17 and42 in 100 cells examined, and had a bimodal distribution with modes setat 32 and 36 (FIG. 2B). Both micro- and macro-chromosomes were found inmetaphase-arrested cells.

Plating Efficiency

The plating efficiency of the GF-1 cells seeded at a density of 100cells/flask was 21% at subculture 50 which increased to 80% atsubculture 80. In comparison, the plating efficiencies of CF, BPS-1,TO-2 and EK cell lines seeded at a density of 100 cells/flask were 22%,13%, 48%, and 63%, respectively. The increase in plating efficiency inGF-1 cells suggests the occurrence of transformation during subcultures50-80.

Effects of FBS Concentration and Temperature on the Growth of the GF-1Cells

FIG. 3 illustrates the effects of FBS concentration on the growth of theGF-1 cells at subcultures 50 and 80. The growth of the GF-1 cells atboth subcultures 50 and 80 corresponded to the concentration of FBS,i.e., the higher the FBS concentration, the greater the growth of cells.However, when the growth rates of the GF-1 cells at subcultures 50 and80 were compared, the GF-1 cells at subculture 80 demonstrated a muchgreater growth potential than those at subculture 50, especially whenthe FBS concentrations were at 2%, 5%, and 10%. For example, at day 4 ofthe cell cultures containing 10% of FBS, the GF-1 cells at subculture 50have 3.5×10⁶ cells/25 cm² flask, whereas the GF-1 cells at subculture 80have 5.0×10⁶ cells/25 cm² flask. These results suggest that therequirement of FBS for cell growth decreased at subculture 80, which isan indication that the transformation of cells had occurred during theperiod from subculture 50 to subculture 80.

FIG. 4 illustrates the effect of temperature on the growth of the GF-1cells at subculture 80. The results show that the GF-1 cells grew wellat 28° C. and 35° C. However, the growth of the GF-1 cells cultured at35° C. started to decline at day 4, suggesting that maintaining the cellculture at 35° C. may have long-term effects on cell growth. The GF-1cells did not grow well at 18° C.

In accordance with a second embodiment of the present invention, thereare provided methods for producing the aquatic viruses in the GF-1cells, purifying the viruses, and detecting a virus in the immortal cellline. The experimental designs pertaining to this embodiment areillustrated as follows:

EXAMPLE 2 Methods For Producing Viruses Using the GF-1 Cell Line andMethods for Detecting the Viruses in the Cell Line

(1) Test for Susceptibility of the GF-1 Cells to Aquatic Viruses

Infectious pancreatic necrosis virus (IPNV, strain AB, SP, VR299 andEVE), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) andnervous necrosis virus (NNV, GNNV isolate) were used to infect the GF-1cells at subculture 80. The susceptibility to GNNV was also examined inBGF-1 cell line, which was derived from the fin of the banded grouperEpinephelus awoara.

Each of the monolayer GF-1 cells was inoculated with 0.5 ml of variousaquatic virus with titer of 10³ TCID₅₀ /0.1 ml. After a 30-minadsorption period, the cells from each flask were washed three timeswith PBS, followed by the addition of 5 ml of L15-2% FBS to each flask.The flasks were then incubated separately at 20° C. and 28° C. Thesupernatants of culture cells were collected and titrated for 6 dayspost viral infection.

(2) Multiplication and Purification of Aquatic Viruses in the GE-1 CellLine

Viral isolate was inoculated at an MOI (multiplicity of infection) of0.01 into the GF-1 cell line. When CPE appeared, the GF-1 cells werescraped into the medium and the cell debris was pelleted at 10000×g for30 min (the first pellet). The supernatant was transferred to a bottleand polyethylene glycol (PEG, molecular weight 20000) and NaCl wereadded to reach a final concentration of 5% and 2.2% separately. Thesupernatant was then stirred for 4-6 hours at 4° C., and the virusparticles were pelleted by centrifugation at 10000×g for 1 hour (thesecond pellet). The first pellet and the second pellet were re-suspendedin a small amount of TNE buffer (0.1M Tris, 0.1M NaCl, 1 mM EDTA, pH7.3), to which an equal volume of Freon 113 was added. The mixture wasshaken vigorously for 5 min, and the emulsion was separated into theFreon and aqueous phase by centrifugation at 3000×g 10 min. The aqueousphase was collected, layered on a preformed 10-40% (w/w) CsCl gradient,and centrifuged at 160000×g for 20 hours. The visible virus band wascollected, diluted with 10 ml of TNE buffer, and pelleted again bycentrifugation at 150,000 g for 1 hours. The final pellet wasresuspended in a small volume of TE buffer (0.1 M Tris, 1 mM EDTA, pH7.3).

(3) Detection of Aquatic Viruses in the GF-1 Cell Line

In general, when a virus infects a cell line which is susceptible to thevirus, a CPE of the cell culture can be observed within a couple of daysafter the infection. The appearance of CPE serves as evidence that thevirus has successfully infected and multiplied in the cell line. Theviral infection in the cell line can be further confirmed using anelectron microscopic technique which is described as follows: Thevirus-infected cells were fixed in 2.5% glutaraldehyde in 0.1M ofphosphate buffer at pH 7.4 and post-fixed in 1% of osmium tetraoxide.The cells were ultrathin sectioned. The ultrathin sections were stainedwith uranyl acetate-lead citrate and examined under a Hitachi H-600Aelectron microscope. The viral particles should appear as homogeneous,spherical particles in the cytoplasm of the cells.

There are also three methods which are directed to specific detection ofNNV in the GF-1 cell line:

(A) Detection of NNV in the GF-1 Cells by Polymerase Chain Reaction(PCR) Amplification

A PCR amplification method was used to confirm that the GF-1 cells areable to proliferate NNV. The method required that the viral RNA beextracted from the supernatant of the NNV-infected cells after CPEappeared using a Rneasy™ mini kit (QIAGEN). For reverse transcription,extracted viral RNA was incubated at 42° C. for 30 min in 40 □l of2.5×PCR buffer (25 mM of Tris-HCl, pH 8.8, 3.75 mM of MgCl₂, 125 mM ofKCl, and 0.25% of Triton X-100) containing 2 U of MMLV reversetranscriptase (Promega), 0.4 U of RNsin (Promega), 0.25 mM of dNTP, and0.5 □M of the reverse primer R3 (5′ CGAGTCAACACGGGTGAAGA 3′) (SEQ ID NO.1). Following the cDNA synthesis, 40 □l of the cDNA mixture were diluted2.5-fold with diethyl pyrocarbonate (DEPC)-treated H₂O (containing 0.025U of DNA polymerase [Biometra], 0.1 mM of dNTP and 0.5 □M of the forwardprimer F2 [5′ CGTGTCAGTCATGTGTCGCT 3′] [SEQ ID NO.2]), and incubated inan automatic thermal cycler (TouchDown™ thermal cycler, Hybaid company).The target region for the primer set (F2, R3) is T4 (400 bp). The PCRproducts corresponding to T2 and T4 were amplified from the nucleicacids of NNV-infected GF-1 cells.

(B) Detection of NNV in the GF-1 Cells by Western Immunoblot

A western immunoblot method was used to specifically detect the NNVproteins. The viral sample was prepared as follows: NNV was inoculatedinto the GF-1 cells and incubated at 20-32° C. After 5 days ofincubation, the NNV-infected cells were pelleted by centrifugation at1000 g for 10 min. The cell pellets were loaded onto a 10%SDS-polyacrylamide gel. After electrophoresis, the proteins were blottedto an immobilon-P transfer membrane (Millipore), which was then soakedin a 3% skim milk tris buffered saline (TBS) for 1 hr. The membrane wasthen incubated with an antiserum against NNV for 1 hr at roomtemperature, washed with TBS, reacted with a peroxidase-conjugate goatsystem for 1 hr, and stained with a substrate containing 6 mg of4-chloronaphthol in 20 ml of methanol and 60 □l of H₂O₂ in 100 ml ofTBS.

(C) Detection of NNV in the GF-1 Cells by Enzyme-Linked ImmunoabsorbentAssay (ELISA)

ELISA is an immunological method which uses an enzyme-labeledimmunoreactant (antigen or antibody) and an immunosorbent (antigen orantibody bound to a solid) to identify specific serum or tissueantibodies or antigens. The ELISA test was conducted as follows: aneffective amount of purified NNV proteins was coated onto a microtiterplate at 4° C. overnight. Then, 3% of bovine serum albumin (BSA) wasadded to the plate (used as blocking agent) and incubated at 37° C. for1 hr. The plate was then washed 3 times with buffer. Next, a dilutedrabbit anti-NNV serum was added to the plate and incubated at 37° C. for1 hr. This was followed by the addition of goat anti-rabbitIgG-horseradish peroxidase serum at 37° C. for 1 hr and3,3′,5,5′-tetramethyl benzidine was added for color development. Thecolor reaction was stopped with 1 N H₂SO₄. The optical density of thewells in the microtiter plate was measured at 450 nm with an ELISAreader (Dynatech MR 5000).

(D) Detection of NNV in the GF-1 Cells by Immunofluorescent Staining

To detect the virus that proliferated in the GF-1 cells, cell cultureswere fixed by 10% formalin for 12 hrs after viral infection. The fixedcell cultures were treated with 0.2% of Triton X-100 and washed withPBST (phosphate buffer with 0.05% Tween 20). The Triton-treated cellcultures were further washed with 3% of skim milk as blocking agent andthen reacted with mouse anti-NNV serum. Finally, the antibody-treatedcell cultures were stained with fluorescein isothiocyanate (FITC)conjugated goat anti-mouse antibodies.

Results

Table 1 summarizes the results of virus susceptibilities of the GF-1cells to IPNV (AB, SP, VR299, EVE strains), HCRV, EHVF and NNV (GNNVisolate), which were determined by first observing the appearance of CPEin the cells after the viral inoculation, followed by the determinationof viral titers (TCID₅₀/ml).

TABLE 1 Viral Susceptibilities of GF-1 Cells at Subculture 80 InitialViral Virus Yield/ml Cell Inoculum CPE (TCID₅₀/ml) line Virus (TCID₅₀)28° C. 20° C. 28° C. 20° C. GF IPNV AB 10³ − + ND 10^(9.5) SP 10³ − + ND 10^(10.8) VR299 10³ − + ND 10^(9.8) EVE 10³ − + ND 10^(9.6) HCRV 10³− + ND  10^(11.0) EHVF 10³ + + 10^(8.1) 10^(7.0) GNNV 10³ + − 10^(8.3)ND ND: Not done. +: Cytopathic effect (CPE) was observed.

As shown in Table 1, for the IPNV strains and HCRV, CPE appear only whenthe cells are incubated at 20° C. For EHVF, CPE appears at both 20° C.and 28° C. However, for GNNV, CPE appears at 28° C. The yields of theviruses in GF-1 cells at subculture 80, which are ranged between10^(7.0) (as for EHVF at 20° C.) and 10^(11.0) (as for HCRV at 20° C.),are extremely high.

Typically, for an aquatic virus such as GNNV, CPE began at the 3rd dayof infection when some rounded, granular, refractile cells began toappear in the cell culture (FIG. 5). Soon more and more cells becameround and swollen. The swollen cells became larger and finally startedto detach from the cell culture and float in the culture media. Most ofthe detached cells were completely disintegrated. The culture fluid fromcell culture showing CPE could transmit other GF-1 cells. Thisexperiment also tested the susceptibility of BGF-1 cell line (derivedfrom the fin of the banded grouper Epinephelus awoara) to GNNV. Theresults showed that no CPE was found after the viral infection.

Typically, for an aquatic virus such as GNNV, the virus could beobserved in the cytoplasm of the GF-1 cells under electron microscope asnumerous non-enveloped, homogeneous, spherical to icosahedral particleswith diameter of 20-25 nm (FIG. 7). Some of the viral particles wereincluded in the inclusion bodies and the others could be found in thecytoplasm (FIG. 7). The isolated viral particles could be furtherpurified by CsCl density gradient centrifugation. Using GNNV as anexample, the purified virus was a non-enveloped icosahedral virionparticle with the diameter of 20-25 nm. The buoyant density of GNNV inCsCl was 1.34 g/cm³.

In addition to the findings of CPE in the GF-1 cells, the existence ofan aquatic virus in the GF-1 cells and the capability of the cells tomultiply the virus can be further confirmed by four methods: (1) the PCRmethod; (2) the Western immunoblot method; (3) the ELISA method; and (4)the immunofluorescent staining method.

Using GNNV as an example, the PCR method could be accomplished bychoosing a pair of primers, i.e., R3 (SEQ ID NO. 1) and F2 (SEQ IDNO.2), for PCR amplification. The target fragment T4 exists in fishnodavirus. Therefore, the PCR method using F2 and R3 was specific tofish nodavirus, not just GNNV. The results of the PCR study showed thatGNNV could be replicated in the GF-1 cells and released into thesupernatant of culture cells (FIG. 6).

The Western immunoblot using mouse anti-GNNV serum demonstrated thatviral proteins were present in the GNNV-infected cells cultured at20-32° C., suggesting that the viral mRNA could be successfullytranslated into viral polypeptides within the host cells when theculture was maintained at 20-32° C.

The ELISA and immunofluorescent staining methods also showed positivereactions with the anti-GNNV serum, indicating that GNNV could bemultiplied in the GF-1 cells.

In accordance with a third embodiment of the present invention, there isprovided for an anti-NNV antibody and the method of making the antibody.The experimental designs in this embodiment are illustrated as follows:

EXAMPLE 3 Production of Anti-NNV Antibodies

(1) Production of Anti-NNV Antibodies

Polyclonal antibodies can be produced in accordance with conventionalmethods, e.g., by sequential injections of the purified NNV immunogeninto a suitable animal such as a rabbit, rat, or mouse. For example, asuitable amount of the NNV immunogen can be injected intravenously,subcutaneously, or intraperitoneally to a rabbit and boosted twice ormore at 2 or 3 week intervals. The injection may contain a suitableamount of Freund's complete or incomplete adjuvant, if necessary.

For the production of monoclonal antibodies, immunizing mice ispreferred. Three or four days after the final boost, spleen cells ofmice can be separated and fused with myeloma cells, e.g., SP2/0-Ag14myeloma cells (ATCC CRL 1581), in accordance with a conventional methoddescribed by Mishell and Shiigi (Selected Mthods in Cellular Immunology,W. H. Freeman & Company, 1980). The spleen cells and the myeloma cellscan be used in a ratio ranging from 1:1 to 1:4. A fusion-promotingagent, e.g., polyethylene glycol (PEG) 4000, may be employed foraccelerating the cell fusion. A medium suitable for use in the cellfusion step may be RPMI 1640 (Gibco BRL, Life Technologies, Inc.) andthe medium generally contains 10-15% (v/v) fetal bovine serum (FBS).

The fused cells can be cultured in the RPMI1640-15% FBS, supplementedwith hypoxanthine, thymidine and aminopterin, and after seven to tendays, positive hybridoma clones producing antibodies specific for NNVcan be selected by ELISA assay using the culture supernatant. Furtherselection of positive clones can be accomplished by using conventionalmethods, e.g., the limiting dilution technique, the plaque method, spotmethod, agglutination assay and autoradiographic immunoassay.

(2) Purification of Antibodies

Antibody can be purified by conventional immunoglobulin purificationprocedures such as ammonium sulfate precipitation, gel electrophoresis,dialysis, affinity chromatography, and ultrafiltration. Ion exchange,size exclusion hydroxylapatite, or hydrophobic interactionchromatography can be employed, either alone or in combination. Lightand heavy chain can be carried out using gel electrophoretic techniquesor isoelectric focusing, as well as other techniques known in the art.

In accordance with a fourth embodiment of the present invention, thereis provided a vaccine to NNV and a method for protecting fish againstNNV infection. The experimental designs in this embodiment areillustrated as follows:

EXAMPLE 4 Production of NNV Vaccines

Preparation of Vaccine using Killed NNV

The vaccine of the present invention is administered as a killedvaccine, which encompasses any methods now known or hereafter developedfor killing. The preferable method is by heat treatment. The heattreatment method can be accomplished by heating the purified NNV to atemperature sufficiently to inactivate the virus (such as 70° C.) for asufficient amount of time (such as for 24 hours). After the heating stephas been completed, for intraperitoneal or intramuscular vaccination,the inactivated NNV can be emulsified in Freund's incomplete adjuvant(FIA) using a mixer for several minutes. The vaccine can then beinjected into the fish (the primary injection). Booster injections canbe given to the fish 30-45 days after the primary injection. Normally,the booster injection consists of about one half of the volume of thevaccine used in the primary injection. The fish then can receive asecondary boost 10 days after the first booster shot is administered.The serum samples from the fish at various time points can be taken fortiter determination.

For orally administered vaccine, an enteric coating containing non-toxicpolymeric materials can be added to the vaccine. The preferable entericcoating materials are the ones which can resist dissolution at the pH ofthe stomach but can be dissolved once the material passes from thestomach to the pyloric caecum and intestines. For example, celluloseacetate phthalate, hydroxypropylmethyl cellulose phthalate,carboxymethylethyl cellulose, hydroxypropylmethyl cellulose acetatesuccinate, cellulose acetate trimellitate, polyvinyl acetate phthalate,EUDAGRIT L-30D and 1100-55, EUDAGRIT L 12.5 and L 100, EUDRAGIT E, RL,RS and NE are among the preferred materials. Additional materials can beused in combination with the enteric coating materials. For instance,plasticizers (such as polyethylene glycol 200, 400, 1000, 4000, 6000,propylene glycol, PVPK-90, glycerin or glycerol, diethyl phthalate,oleic acid, isopropyl myristate, liquid paraffin or mineral oil,triacetin, glycerol monostearate, dibutyl sebacate, triethyl citrate,tributyl citrate, acetylated monoglyceride, dibutyl phthalate, acetyltributyl citrate, castor oil, and glycerol tributyrate); disintegrants(such as sodium starch glycolate); adjuvants (such as immunostimulants[e.g., beta glucan]); binders (such as starch, polyvinyl pyrrolidone,polyvinyl alcohol); diluents (such as lactose); lubricants (such asmagnesium stearate) etc. can all be used with the enteric coatings. Fororal administration, fish can receive the vaccine on an every-other-daybasis for a total of thirty days. The effects of the vaccines can bemonitored by the use of ELISA.

Orally administered vaccine is generally the preferred method ofvaccinating fish because it is not limited by the size of the fish thatcan be handled, and it reduces the stress on the fish associated withimmersion and intraperitoneal injection. Furthermore, oral vaccinesoffer the additional advantages of stimulating the gut-associatedlymphoid tissue to a greater extent than does intraperitoneal injection.

The present invention has been described with reference to severalpreferred embodiments. Other embodiments of the invention will beapparent to those skilled in the art from the consideration of thisspecification or practice of the invention disclosed herein. It isintended that the specification and examples contained herein beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims:

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: primer_bind <222> LOCATION:<223> OTHER INFORMATION: synthetic oligonucleotide pr#imer complementary       to viral sequence<300> PUBLICATION INFORMATION: <400> SEQUENCE: 1cgagtcaaca cgggtgaaga             #                  #                   # 20 <210> SEQ ID NO 2 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: primer_bind <222> LOCATION:<223> OTHER INFORMATION: synthetic oligonucleotide pr#imer complementary       to viral sequence<300> PUBLICATION INFORMATION: <400> SEQUENCE: 2cgtgtcagtc atgtgtcgct             #                  #                   # 20

What is claimed is:
 1. A method for growing a virus comprising:inoculating said virus into an established cell culture comprising animmortal cell line which is ATCC deposit no. PTA-859; and incubatingsaid cell culture in a nutrient medium suitable for growth andreplication of said virus.
 2. The method for growing a virus accordingto claim 1, wherein said virus is selected from the group consisting ofBirnaviridae, Herpesviridae, Reoviridae, and Nodaviridae.
 3. The methodfor growing a virus according to claim 2, wherein said Birnaviridae isInfectious Pancreatic Necrosis Virus (IPNV).
 4. The method for growing avirus according to claim 2, wherein said Herpesviridae is Eel HerpesVirus Formosa (EHVF).
 5. The method for growing a virus according toclaim 2, wherein said Reoviridae is Hard Clam Reovirus (HCRV).
 6. Themethod for growing a virus according to claim 2, wherein saidNodaviridae is a fish nodavirus which is selected from the groupconsisting of Nervous Necrosis Virus (NNV), Fish Encephalitis Virus(EFV), Piscine Neuropathy Nodavirus (PNN), Grouper Nervous NecrosisVirus (GNNV), Stripped Jack Nervous Necrosis Virus (SJNNV), Tiger PufferNervous Necrosis Virus (TPNNV), Berfin Flounder Nervous Necrosis Virus(BFNNV) and Red Spotted Grouper Nervous Necrosis Virus (RGNNV).
 7. Amethod for mass producing a virus comprising: inoculating the virus inthe established cell culture comprising an immortal cell line which isATCC deposit no. PTA-859; incubating said cell culture in a nutrientmedium suitable for growth and replication of said virus until anappearance of cytopathic effects (CPE); and harvesting said virus fromsaid cell line.
 8. A method for purifying a virus comprising:inoculating the virus into an established cell culture comprising animmortal cell line which is ATCC deposit no. PTA-859; incubating saidcell culture in a nutrient medium suitable for growth and replication ofsaid virus until an appearance of cytopathic effects (CPE); harvestingsaid virus from said cell line; and purifying said virus using a densitygradient centrifugation.
 9. The method for purifying a virus accordingto claim 8, wherein said density gradient centrifugation is a CsCldensity gradient centrifugation.
 10. The method for purifying a virusaccording to claim 8, wherein said virus is NNV, and wherein said NNVhas a buoyant density of approximately 1.34 g/ml in CsCl.